Heating mechanism for dna amplification, extraction or sterilization using photo-thermal nanoparticles

ABSTRACT

A heating mechanism for use in DNA applications such as DNA amplification, extraction and sterilization is provided. Nanoparticles having photo-thermal properties are put in contact with a reaction mixture and irradiated with an activation light beam to activate these photo-thermal properties, thereby releasing heat. Nanoparticles of several types may be used. Use of the same nanoparticles or of different one to monitor the reaction using a different light beam is also presented.

RELATED PATENT APPLICATION

This application claims priority to U.S. provisional patent applicationSer. No. 61/737,175, filed on Dec. 14, 2012, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present description relates to processes involving DNA and moreparticularly concerns a heating method for performing such molecularbiological techniques using nanoparticles having photo-thermalproperties.

BACKGROUND

Polymerase Chain Reaction (PCR) is a DNA amplification technique whichis essential to genetics, and particularly in next generationsequencing, where amplification of the quantity of starting DNA iscommonly performed. It is an example where technology and basic researchhave been combined to deliver a tool that has been applied to amultitude of fields such as genomics, forensics, DNA/RNA aptamersoptimization, and diagnostic testing.

PCR is a temperature mediated process that requires cycling between settemperatures. Single strand DNA is required for two primer sequences tobind upstream and downstream of the region to be amplified. To allowthis to occur, the first step is denaturation or separation of the twostrands at around 94-98° C. Primer annealing occurs around 45-55° C. andallows the thermo-stable polymerase to bind to defined regions of doublestranded DNA. The next stage is elongation of the double stranded copywhere the temperature is raised to the optimum temperature (around 72°C.) for the enzyme catalysis to proceed. Finally, temperature isreturned to 94° C. for denaturation to single stranded DNA that allowsthe cycle repeat.

The thermal cycler (also known as a Thermocycler, PCR Machine or DNAAmplifier) is an apparatus used to amplify segments of DNA via thepolymerase chain reaction (PCR) process. Thermal cyclers are typicallyprovided with a thermal block with holes where tubes holding the PCRreaction mixtures can be inserted. Heat is provided through solid stateheaters or infrared lamps. The cycler raises and lowers the temperatureof the thermal block in discrete, preprogrammed steps.

There is a need if the field to increase the speed, and therefore theefficiency, of PCR processes. The duration of the thermocycling of a PCRprocess can be dependent upon several factors, including theexperimentalist's requirements. Indeed, for a molecular biologistinvolved in sequencing large sections of a genome, amplification oflarge fragments would require a longer cycle time than in for morecommonplace diagnostic applications, for example, to ensure high yield.The additional time required is a function of the temperature ramp timeand cooling between the stages of PCR (Denaturation, primer annealingand elongation/synthesis). Shortening ramp and cooling times means morerapid transition and shorter cycling times, even appreciating for longfragments, a more substantial pause at the elongation temperature isrequired reflecting the polymerisation rate of the enzyme, expressed inbase pairs per second (ranging from a few hundred to 1 kilobase persecond). The cycle time can be shortened with more rapid enzymes or byallowing incomplete amplification of amplicons that are termedmega-primers to be completed in subsequent cycles. Though the latertechnique lowers the overall yield from 30 cycles it does allow slowerpolymerases to be utilised. More fundamental is that very fewinstruments on the market are available of delivering cycle times ofless than 7 minutes, to full exploit rapid cycling and at a costsuitable to wide spread application.

An example of such thermocycler is the Lightcycler® that has beencommercialized by Roche. The Lightcycler® can achieve heating rates of15° C. per second with cooling rates of 10° C. per second, but commonlyramp times are significantly less than this, at around 2-5° C. persecond (heating), reflecting heat delivery by Peltier elements thatstruggle to produce rapid heating of aluminium or ceramic blocks used tohold tubes.

Another downside of commercial real time quantitative thermal cyclersknown in the art is the cost of each instrument, running into tens ofthousands of dollars for rapid thermocycling. The current high cost ofall PCR thermocycler platforms (real time PCR inclusive) represents asignificant research cost to the experimentalist. PCR is the backbone ofmany molecular biological studies since its popularization by NobelLaureate Kary Mullis and improvements to both method and instrument arealways sought.

The biological components have been demonstrated to be able to run muchfaster than common instrumental cycle times. It would therefore beadvantageous to provide an instrument which scales and lowers the costburden such that its use becomes more widespread, while still deliveringsub 10 minute reaction times for 30 cycles.

Other DNA amplifications are known in the art. One example isLoop-mediated isothermal amplification (LAMP), which involves holding atemperature (for example 65° C.) to allow Bst enzymes to perform a loopamplification using specially designed primers, to cause the formationof one massive repeating chain DNA extended polymer. Although rampingand cycling times are less of an issue, it is still desirable to providean efficient and economical means to control the temperature of thereaction. The same can be said for any DNA amplification technique whereheat needs to be applied to the reaction mixture.

Heating a reaction mixture that contains a DNA molecule is not onlyuseful for DNA extraction techniques but is also used for otherDNA-involving processes, such as cell lysis and sample sterilization.

There is therefore a need for a heating method and device for reactionmixtures containing DNA which alleviates at least some of theaforementioned drawbacks.

SUMMARY

In accordance with one aspect of the invention, there is provided amethod of heating a reaction mixture containing a DNA molecule. Themethod includes the steps of:

-   -   contacting the reaction mixture with nanoparticles having        photo-thermal properties;    -   irradiating the nanoparticles using an activation light beam        activating said photo-thermal properties, such that said        nanoparticles release heat sufficient to provide said heating.

Use of such a method for amplifying the DNA molecule, extracting the DNAmolecule from a prokaryotic or eukaryotic entity or for sterilizing thereaction mixture is also provided.

In one variant, there is provided a method of amplifying a DNA templatecomprising at least one thermal cycle comprising heating a reactionmixture containing the DNA template, each of the at least one thermalcycle comprising the steps of:

-   -   contacting the reaction mixture with nanoparticles having        photo-thermal properties;    -   irradiating the nanoparticles using an activation light beam        activating said photo-thermal properties, such that said        nanoparticles release heat sufficient to provide elongation and        denaturation of said DNA template.

In another variant, a method of extracting a DNA molecule from aprokaryotic or eukaryotic entity is provided, comprising heating areaction mixture containing the prokaryotic or eukaryotic entity, saidheating comprising the steps of:

-   -   contacting the reaction mixture with nanoparticles having        photo-thermal properties;    -   irradiating the nanoparticles using an activation light beam        activating said photo-thermal properties, such that said        nanoparticles release heat sufficient to allow extraction of the        DNA molecule from the prokaryotic or eukaryotic entity.

In yet another variant there is provided a method of sanitizing areaction mixture comprising a DNA molecule comprising heating thereaction mixture said heating comprising the steps of:

-   -   contacting the reaction mixture with nanoparticles having        photo-thermal properties;    -   irradiating the nanoparticles using an activation light beam        activating said photo-thermal properties, such that said        nanoparticles release heat sufficient to sanitize the reaction        mixture.

In some embodiments, the method of heating a reaction mixture containinga DNA molecule includes a step of monitoring this heating.

In one embodiment, the monitoring involves probing the nanoparticleswith a probing light beam having a wavelength different than awavelength of the activation light beam and coordinated with anabsorption feature of the nanoparticles spectrally separate from thephoto-thermal properties used to release heat. For example, thenanoparticles have an elongated geometry, the wavelength of theactivation light beam is coordinated with a longitudinal resonance ofthe nanoparticles and the wavelength of the probing light beam iscoordinated with a transversal resonance of the nanoparticles.

In another embodiment, the step of monitoring the heating involvescontacting the reaction mixture with probing nanoparticles having anabsorption feature spectrally separate from the photo-thermal propertiesused to release heat, and probing the nanoparticles with a probing lightbeam having a wavelength different than a wavelength of the activationlight beam and coordinated with said absorption feature.

According to another aspect of the invention, there is provided anapparatus comprising a heating module for heating a reaction mixturecontaining a DNA molecule, the heating module comprising:

-   -   a thermal block for receiving the reaction mixture in contact        with nanoparticles having photo-thermal properties;    -   a light generating assembly for irradiating the nanoparticles        using an activation light beam activating said photo-thermal        properties, such that said nanoparticles release heat sufficient        to provide said heating.

Other features and advantages of the invention will be better understoodupon reading of embodiments thereof with reference to the appendeddrawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematized representations of thermal blockproviding indirect (FIG. 1A) and direct (FIG. 1B) contact between areaction mixture and nanoparticles heaters.

FIG. 2 is a schematized representation of an apparatus for DNAamplification according to one embodiment.

FIG. 3 is a graphic representation of the relationship observed betweenthe diameter of the nanoparticles and their efficacy.

FIG. 4 is a graphic representation of the variation of extinction (AU)in view of the wavelength (nm) used.

FIG. 5A is a schematized representation of the use of probenanoparticles for temperature monitoring; FIG. 5B is a diagram of anapparatus for DNA-related applications comprising a heating module and amonitoring assembly according to one embodiment.

FIG. 6 illustrates a thermal cycling demonstration graphic.

FIG. 7 is a photograph of a 1.5% agarose gel demonstrating the formationof product through use of a plasmonic thermocycler.

FIG. 8A is a photograph of an agarose gel and FIGS. 8B and 8C aregraphic representations of the optimization of the nanoparticle contentof a PCR mixture.

FIG. 9 is a photograph of a gel using pre-treatment to determine whetherdamage is occurring to either DNA or enzyme components of the PCRreaction mixture, wherein SM denotes size markers.

FIG. 10 is a photograph of PCR products obtained by plasmonic PCR withnanoparticles in the PCR mixture, wherein lane 1: size markers; lane 2:negative control; lane 3: plasmonic PCR product (10 μl loading); lane 4:plasmonic PCR product (20 μl loading); lane 5: plasmonic PCR product (20μl loading); lane 6: positive control (conventional PCR); and lane 7:size markers.

FIG. 11 is a graphic representation of a thermal trace of 30 rapidcycles.

FIG. 12 is a graphic representation of a contact PCR experiment tracecycling between 91° C. (denaturation), 55° C. (annealing), and 72° C.(elongation), wherein the spike present when transitioning from 55° C.to 72° C. and the jumps from 72° C. to around 80° C. are not physicaland are due to problems with the instrument readout.

FIG. 13 illustrates the heating and cooling rates obtained from rapidtemperature cycling, wherein solid lines denote average values anddashed lines are located one (sample) standard deviation away fromaverages.

FIGS. 14A to 14C are histograms showing the temperature variation of thePCR solution at the temperatures: 91° C., 55° C. and 72° C.,respectively, wherein the solid grey lines are placed at the targettemperatures; the darker solid lines are located at the averagetemperatures; and the dashed lines are located one (sample) standarddeviation away from the corresponding average.

DESCRIPTION OF EMBODIMENTS

In accordance with one aspect of the present description, there isprovided a method of heating a reaction mixture containing a DNAmolecule. As will be understood from the description below, embodimentsof the invention provide a heating mechanism for use in DNA applicationssuch as DNA amplification, extraction and sterilization, through theirradiation of nanoparticles having photo-thermal properties.

Embodiments of the invention may be applied to any DNA related processwhere heating of a DNA template is required. It will be understood thatreferences to DNA are also meant to encompass RNA related embodiments.

In some embodiments, the DNA process may be a DNA amplification processsuch as, for example, is a polymerase chain reaction (PCR) process. Asmentioned above, PCR is a temperature mediated process that requirescycling between set temperatures. It therefore involves a thermalcycling of the reaction mixture through multiple heating and coolingstages. For such applications, the reaction mixture typically includes aDNA template, a primer capable of annealing to the template and athermostable polymerase. Single strand DNA is required for two primersequences to bind upstream and downstream of the region to be amplified.To allow this to occur, the first step of the PCR process isdenaturation or separation of the two strands of the DNA template, whichtypically occurs around 94-98° C. Primer annealing then occurs around45-55° C. and allows the thermo-stable polymerase to bind to definedregions of double stranded DNA. The next stage is elongation of thedouble stranded copy where the temperature is raised to the optimumtemperature for the enzyme catalysis to proceed, topically around 72° C.Finally, temperature is returned to 94° C. for denaturation to singlestranded DNA, allowing the cycle to be repeated. This cycle is repeateda number of times, typically 20 to 40 cycles.

In another embodiment, the DNA amplification may be a Loop-mediatedisothermal amplification (LAMP). In LAMP, the target DNA sequence isamplified at a constant temperature, typically around 65° C. Usingspecially designed primers, the formation of one massive repeating chainDNA amplificons/extended polymer is obtained. LAMP alleviates the needfor thermal cyclers, but still requires suitable heating capabilitiesand monitoring mechanisms.

Other examples of DNA amplification processes include recombinaseamplification, helicase amplification, whole genome amplification or anytechnique using polymerase enzymes and generally requiring one or moreheating steps.

In other embodiments, the heating method described herein may be used aspart of a DNA extraction or isolation process. There is thereforeprovided a method for extracting DNA from a prokaryotic or eukaryoticentity such as a cell, virus or bacteria. Such processes may for examplerequire performing cell lysis on a DNA sample, which may be performed byheating the cell to a predetermined temperature. In such embodiments,the expression “prokaryotic or eukaryotic entity” is understood todescribe living entities as well as any DNA/RNA containing non-liveentities such as phage, viruses and retroviruses.

In other embodiments, the heating method may be applied to DNAsterilization processes where heat is applied to kill bacteria in a DNAsample prior to further processing of this sample such as extraction oramplification.

Although the examples below refer mostly to PCR, it will be readilyunderstood, therefore that variants could be applied to other techniqueswhere heat is to be applied to at least a portion of a DNA relatedprocess, without departing from the scope of the present invention.

The expression DNA molecule is used herein to describe a DNA molecule ofinterest to a process such as amplification and extraction or any othermolecule part of a mixture requiring a heating step.

The expression “reaction mixture” is meant to refer to the ensemble ofcomponents required for the DNA process. In embodiments related to PCRapplications, the reaction mixture may include:

-   -   The DNA template that contains the DNA target molecule to be        amplified. In examples of application, the DNA template may be        from genomic (human, bacterial, viral), fragmented (forensic and        archaeological samples), plasmid or mitochondrial.    -   At least one primer. For typical PCR applications, two primers        that are complementary to the 3′ ends of each of the sense and        anti-sense strand of the DNA target are generally provided.    -   A thermostable polymerase. The best known DNA polymerase used        for PCR is the Taq polymerase but other types of DNA polymerase        may also be used such as polymerase purified from other        thermophilic microbes; computationally designed enzymes that        could be modifications of either TAQ or other polymerases.    -   Additional reactants, including, non-exhaustively, nucleotides        containing triphosphate groups such as Deoxynucleoside        triphosphates, the building-blocks from which the DNA polymerase        synthesizes a new DNA strand; Divalent cations, magnesium or        manganese ions; and monovalent cation potassium ions.    -   A buffer solution which providing a suitable chemical        environment for optimum activity and stability of the DNA        polymerase.

Heating Mechanism and Nanoparticles

The method of heating a DNA template according to embodiments of theinvention generally includes the steps of contacting the reactionmixture containing the DNA template with nanoparticles havingphoto-thermal properties, and irradiating the nanoparticles using anactivation light beam activating these photo-thermal properties, suchthat the nanoparticles release heat sufficient to provide the desiredheating. In effect, instead of using Peltier heaters or infrared lampsto transfer heat to the vessel containing the reaction mixture,embodiments of the invention use nanoparticles as “heaters”.

The nanoparticles may be embodied by any particles of nanometricdimensions capable to release heat upon optical stimulation.Nanometer-sized particles are often defined as particles with at leastone dimension below 100 nm. Particles not meeting this threshold, butstill of a small enough size to exhibit properties typically associatedwith nanoparticles, may however still be considered within the scope ofthe present invention. The nanoparticles may for example be embodied bynanospheres or nanorods made of a metal such as gold, silver or thelike, carbon nanotubes coated with a metal or multiwalled carbonnanotubes coated with or decorated with a metal, and the like. The metalmay for example be Gold (Au), Silver (Ag), Palladium (Pd), Platinum(Pt), Iron (Fe), Copper (Cu), Aluminum (Al), Zinc (Zn) or the like. Boththe dimensions and geometry of a given type of nanoparticles may have animpact on the associated heating efficiency.

The expression “photo-thermal properties” is meant to refer to theability of a given type of nanoparticles to release heat as a result ofan optical stimulation, i.e. the irradiation of these nanoparticles witha light beam having suitable optical characteristics. The photo-thermalproperties may result from various chemical, geometrical or physicalcharacteristics of the nanoparticles.

In one embodiment, the photo-thermal properties include a localizedplasmon resonance at a surface of the nanoparticles, resulting in a“plasmonic heating” effect. This effect is for example observed at thesurface of gold nanoparticles, spherical or having another geometry.Plasmonic heating may also be observed un gold coated or decoratedmultiwalled carbon nanotubes. A localized surface plasmon originatesfrom a strong interaction between gold or silver nanoparticles andexcitation light having a wavelength which resonates with the surfaceplasmon. Under excitation by light at the resonance wavelength apolarized charge build up at the surface of the particle leads to anoscillating dipole around the particle that exhibits an enhancedabsorption and scattering cross section. The resonant wavelength isdetermined by the size and geometry of the nanoparticles. The energy ofthe resonance of the oscillating dipole is dispersed through Ohmicheating losses to the surrounding medium, raising its temperature. Theenergy released can be used to heat a solution rapidly where eachparticle becomes a heating element.

In other embodiments, the photo-thermal properties may be any process byin which energy from light is absorbed by a nanoparticle, leading to aneventual decay resulting in conversion to heat energy to the surroundingmedia. Nanoparticles may be mono-dispersion in solution, in direct orindirect contact with reaction components or immobilised within apolymeric material or glass.

The contact between the reaction mixture and the nanoparticles may bedirect or indirect. Embodiments showing both types of contacts are shownin FIGS. 1A and 1B, respectively. In both case, a thermal block 20 wherethe thermal cycling of the reaction mixture occurs is shown. Referringmore particularly to FIG. 1A, in the illustrated embodiment the thermalblock 20 includes an inner vessel 22 in which is introduced the reactionmixture 24. The inner vessel 22 is inserted in a larger outer vessel 26.The nanoparticles 28, in this embodiment in nanofluid form, areintroduced in the outer vessel 26. Both the inner and outer vessels 22and 26 may be embodied by any appropriate structure such as tubes,capillaries and the like. An activation light beam 30 is used toirradiate the nanoparticles, which therefore release heat according totheir photo-thermal properties. The heat is transferred to the innervessel 22 and to its contents, thereby heating the reaction mixture 24.As the fluids containing the nanoparticles and the reaction mixture arekept physically separate, the contact between the nanoparticles andreaction mixture can be said to be indirect. The embodiment of FIG. 1Bdiffers from that of FIG. 1A in that there is a single vessel 25, inwhich the nanoparticles 28 are provided in solution with the reactionmixture 24. In this case, the contact can be said to be direct. In othervariants, indirect contact could also be defined as an emulsion of amodified nanofluid and water. The expression “contacting the reactionmixture with nanoparticles” is understood to refer to providing eitherdirect contact, indirect contact or both at the same time.

In some embodiments, in particular where the nanoparticles are in directcontact with the reaction mixture, care should be taken so that thenanoparticles do not interfere with the DNA related process to beperformed. For example, in PCR embodiments, the polymerase may bind tothe surface of the nanoparticles, blocking positive active site byassociation with a negatively charge particle surface, and thereforeinhibiting the polymerase from performing its function during the DNAamplification process. In accordance with some embodiments, therefore,the nanoparticles have a surface modification by a chemical compoundsuch as Polyethylene glycol (PEG) or any other chemical equivalent thatprevents the inhibition of a positive active site of polymerase classenzymes.

In one example, 840 pM of uncapped gold nanorods is mixed vigorouslywith an aqueous thiol PEG 5000 MW solutions (1 mg/ml) in equal volumesand incubated at 30° C. for 2 hours (elevated temperature decreasesreaction time for formation of Au-S-PEG complex). The mixture containingthe nanorods is centrifuged down at 13,000 RPM, in order to remove thesupernatant. The nanorods are resuspended in MilliQ water using a vortexand afterwards centrifuge down again to form a highly coloured pellet,with a final resuspension in DNase/RNase free water.

One advantage of the surface modification described above is that theresulting nanoparticles can be used as generic heaters independently ofthe type of polymerase, size of the template or template type.Inhibition prevention will apply to any application, avoiding theadditional complication of limiting the application of the method tospecific polymerase types.

In various embodiments, the dimensions of the nanoparticles may beselected in view of optimizing the resulting heating efficiency.Referring to FIG. 3, there is shown a graph of the heating efficiency ofgold nanorods with respect to their aspect ratio (defined as thelongitudinal axis divided by the diameter in nanometers), for nanorodshaving a diameter of 10 nm and 25 nm. In this comparison it can be seenthat the nanorods of 10 nm of diameter provide a greater heatingefficiency.

In various embodiments, other factors can be controlled to improveheating efficiency of the method. As predicted by the Beer-Lambert law,a higher rod concentration and shorter path-length will maximize theabsorbance of the activation light beam. By way of example, to study theoptimization of the heating process, data was calculated fromcommercially available nanorod solution from Nanopartz (tradename) withno capping ligand. The resonance spectra for the various nanoparticlesstudied is shown in FIG. 4. Referring to Table 1, data showing theoptimization of the resonance of the nanoparticles, path length andconcentration to achieve maximum absorbance is presented. For eachnanoparticle model considered, designated by part number, Table 1 liststhe wavelength if the surface plasmon resonance (SPR), the maximumconcentration of nanoparticles in the solution, and the converted powerfor an optical path length of 0.5 cm.

TABLE 1 Optimization of the resonance of the nanoparticles, path lengthand concentration Converted Power for 0.5 cm Optical Path Length MaximumStock Maximum Concen- Concen- Concen- Part SPR tration tration tration26.3 pM Number (nm) (pM) (%) (%) (%) A11-60 536 6.58 61.5 14.5 45.5A12-10-808 808 57.7 65.3 95.4 95.4 A12N-25-1400 1400 3.70 29.0 42.4 42.4A12N-25-1064 1064 5.64 36.4 53.2 53.2 A12-25-980 980 6.48 39.0 57.0 57.0A12-25-850 850 8.38 44.2 64.5 64.5 A12-25-808 808 9.28 46.1 67.5 67.5A12-40-650 650 6.10 36.0 52.6 52.6 A12-40-700 700 3.22 21.7 31.7 31.7

As seen from Table 1, heating efficiency can be impacted by selection ofthe nanoparticles and concentration parameter. Also, if 95% absorbanceat 808 nm is achieved, path length can be reduced significantly.Concentration used was increased from 26.3 pM to more than 800 pM, whichallows a reduced path length to be applied. Under the Beer-Lambert lawabsorbance increases as concentration increases, allowing a short pathlength through a solution to be used with respect to achieving the sameheating rate as for a low concentration of particles. Furthermore, itallows miniaturisation of sample volume. This allows the method totranslate to bulk heating of water within micro channels. Using thescaling factor of 840 pM/26 pM, this indicates how much it is possibleto increase nanoparticle concentration, by 32.3 times for example. Thisalso means that the path length can be reduced by the same scaling.Shorter path lengths are needed to achieve the same absorbanceeffectively. The path length was reduced to 0.154 mm which is of theorder of the height of a microfluidic channel. The method is thereforeapplicable to microfluidic chips such as the Fluidigm (tradename) systemfor digital PCR.

Apparatus

Referring to FIG. 2, an apparatus 32 for performing DNA extractionaccording to one embodiment is schematically illustrated.

The apparatus first includes a thermal block 20 for receiving thereaction mixture in contact with nanoparticles having photo-thermalproperties. The thermal block may be embodied by any container, chamber,assembly, or other structure adapted to receive the reaction mixture andnanoparticles and provide optical access thereto. As mentioned abovewith respect to FIGS. 1A and 1B, the thermal block may be configures toprovide indirect or direct contact between the reaction mixture andnanoparticles. In the illustrated embodiment, by way of example only,the thermal block 20 is embodied by a glass capillary sized to receivefrom 25 to 40 μl of the solution containing the reaction mixture andnanoparticles. A thermocouple 21 may optionally be used to measure thetemperature change in the vessel containing the nanoparticles.

The apparatus 32 further includes a light source 34 for irradiating thenanoparticles using an activation light beam 30 activating theirphoto-thermal properties, such that the nanoparticles release heatsufficient to provide the desired heating. In one embodiment, the lightsource 34 may be embodied by a laser or LED (light-emitting diode)generating light at a wavelength coordinated with the photo-thermalproperties of the nanoparticles. The light source may be part of a lightgenerating assembly allowing a control of optical parameters of theactivation light beam such as the wavelength, optical power, duty cyclein embodiments where the light beam is pulsed, spot size, etc. Variousmeans of adjusting such parameters are well known in the art and neednot be described here. By way of example, in the illustrated embodiment,the light source 34 generates an activation light beam 30 having awavelength of 532 nm resonant with a plasmon resonance of gold nanorods,and a coil-based shutter 36 is provided in a path of the activationlight beam 30 before it reaches the thermal block 20. The shutter 36 mayfor example be used to periodically block the activation light beam 30to reduce the average light power reaching the thermal block 20. Inother embodiments a different mechanism may be used to control the lightpower such as for example direct modulation of a laser or LED lightsource (such as TTL modulation), use of a modulating device such as anintensity of phase modulator, etc. Of course, one skilled in the artwill readily understand that a number of additional optical componentsmay be provided in the apparatus 32 depending on particular designconsiderations, such as lenses, mirrors, filters, polarisers,amplifiers, and the like without departing from the scope of the presentdescription.

The optical parameters of the activation light beam 30 are preferablydetermined and controlled in view of the photo-thermal properties of thenanoparticles. By way of example, in one embodiment the necessary lightpower to achieve a desired temperature through release of heat from thenanoparticles can be calculated from theoretical considerations relatedto plasmonic heating. The heat released by a given nanoparticle can beevaluated using Equation (1) below, taking for example a sphere-shapednanoparticle, also referred to as a nanosphere. To briefly summarizeequation (1), Δ_(Tmax) is the steady-state surface temperature of thenanosphere relative to the external temperature at distances muchgreater than the dimensions of the nanosphere, ω is the harmonicfrequency of the incident radiation (related to the light wavelength), Ris the nanosphere radius, I₀ is the intensity of the incident radiation,c is the speed of light in vacuum, k₀ is the thermal conductivity of theexternal solution, ∈₀ is the relative complex permittivity of theexternal solution, μ₀ is the relative magnetic permeability of theexternal solution, and ∈_(m) is the relative complex permittivity of thenanosphere:

$\begin{matrix}{{\Delta \; T_{\max}} = {\frac{\omega \; R^{2}I_{0}}{3\; {ck}_{0}\sqrt{ɛ_{0}\mu_{0}}}{\frac{3ɛ_{0}}{{2ɛ_{0}} + ɛ_{m}}}^{2}{{Im}\lbrack ɛ_{m} \rbrack}}} & (1)\end{matrix}$

Assuming that the incident laser beam has a flat-top profile and thatthe reaction mixture is non-absorbing at the wavelength of theactivation light beam, the above relation can be inverted in order tofind the required laser power for a given surface temperature, where Pis the incident laser power and d is the laser spot diameter (equation2).

$\begin{matrix}{P = {\frac{I_{0}\pi \; d^{2}}{4} = {\frac{3\; {ck}_{0}\sqrt{ɛ_{0}\mu_{0}}\Delta \; T_{\max}}{4\omega \; R^{2}}{\frac{{2ɛ_{0}} + ɛ_{m}}{3ɛ_{0}}}^{2}\frac{\pi \; d^{2}}{{Im}\lbrack ɛ_{m} \rbrack}}}} & (2)\end{matrix}$

Time of exposure can also be varied to control the raising of thetemperature of the reaction mixture. As one skilled in the art willreadily understand, the intensity of the activation light beam and thetime of exposure are two parameters which can easily be controlled inconjunction to control the rate at which energy is transferred to thenanoparticles and, consequently, the temperature of the reactionmixture.

The wavelength of the activation light beam is another optical propertywhich can be determined and controlled in view of the photo-thermalproperties of the nanoparticles. As mentioned above, in the case ofplasmonic heating, the release of heat by the nanoparticles results fromtheir stimulation using light having a wavelength matching the localizedplasmon resonance at the surface of the nanoparticles. Furthermore, inembodiments using light-induced plasmonic heating a wavelengthselectable characteristic can be conferred upon the process of heating.Within the bandwidth of excitation, heating of a solution can be turnedon and off readily, accentuated by greater dispersion of heat from thesolution simply by the presence of nanoparticles within the reactionmixture that should, in effective combination with a cooling system,lead to rapid temperature transition, hence shorter PCR cycle times.Carbon nanotubes, in contrast, absorb light in to energy levelspertaining to both the semi-conducting and, if metallic elements arepresent, energy levels pertaining to the presence of the metals. Thebroad absorbance of carbon nanotubes can be explained by many additionaltransitions possible from the ground state over the visible and into thenear infra red for the promotion of an electron. The wavelength of theabsorbance pertains to the difference in energy between the ground stateand the excited state. In any case, by choosing, and optionally varying,the wavelength of the excitation light beam to match a resonance ortransition of the absorption spectrum of the nanoparticles, control ofthe heat released through the photo-thermal properties may be achievedand/or optimized.

Still referring to FIG. 2, the apparatus 32 may include any othercomponent typical of DNA amplification, extraction or sterilizationdevices. For example, in the illustrated example, directed to PCRapplications requiring thermocycling, a fan 38 is provided in proximityto the thermal block 20 and can be activated to accelerate the coolingof the reaction mixture during the cooling phases of the thermocycling.A fan controller 40 preferably allows a control of the activation of thefan 38. Overall control of the apparatus can be managed through anyappropriate device or combination of devices. In the illustratedembodiment, by way of example only, a computer 42 provides electricalcontrol signals to the light source 34 and fan controller 40 through anappropriate electrical interface 44, for example an FPGA circuit board.

Real-Time Monitoring

In accordance with one aspect of the invention, the method may includean additional step of optically monitoring, in real time, thetemperature change resulting from heating a reaction mixture accordingto embodiments of the invention.

Optical properties of nanoparticles can provide a useful spectroscopicapproach for real time monitoring of the reaction. A change in thetemperature of the environment of the nanoparticles also affects thelocal dielectric constant, which leads in a drift of the opticalproperties of the nanoparticles. By using a probe light beam having awavelength coordinated with a different absorption feature than the oneused for heat release, this drift can be measured, therefore monitoringthe corresponding temperature change, by interrogating the nanoparticleresonance with the probe light beam and monitoring a change in eitherthe scattering or absorbance at a fixed probe wavelength.

In some embodiments, probing nanoparticles having an absorption featureat a wavelength different from the wavelength used to activate thephoto-thermal properties of the nanoparticles used for heating can beput in contact with the reaction mixture. Referring to FIG. 5A, there isshown a schematized illustration of the resulting monitoring principle.In the illustrated example, the heating nanoparticles 28 are embodied bynanorods having a localized plasmon resonance as explained above,releasing heat when irradiated with the activation light beam 30 havinga wavelength corresponding to that resonance, for example at about 532nm or 808 nm when considering gold nanorods (bother resonant frequenciescan be changed by particle dimension and the dielectric constant of themedium or surface ligands around them). Probe nanoparticles 46, hereembodied by gold nanospheres, are put in direct contact with thereaction mixture. In the illustrated example of FIG. 5A, the goldnanosphere are shown as covalently attached to the primers and DNAtemplate. Covalent attachment of primers would result in a great localdielectric shift at the surface of the gold nanoparticles as ampliconswould be confined in close proximity to the gold. The plasmonic fieldpropagates approximate tens of nanometers from the surface and changesat the surface more greatly affect the plasmonic shift measured as a redshift of the plasmonic peak. The gold nanospheres used for sensingproduction of amplicons would have a resonance, blue shifted from theresonance of the nanorods used for heating purposes. The probenanoparticles are irradiated with a monitoring light beam 48 having awavelength within the resonance of the gold nanospheres. Return light 50resulting from the interaction of the monitoring light beam with themonitoring nanoparticles is detected and analysed. The intensity of thereturn light varies according to the degree of absorption of themonitoring light beam 48 by the gold nanospheres. As the temperaturevaries, the resonance of the monitoring nanoparticles shifts, and thewavelength of the monitoring light beam falls in and out of resonance,changing the degree at which the monitoring light beam is absorbed.

In one example, gold probe nanoparticles are covalently linked to theprimers through a thiol linkage added to the 5′ end of the primer andlinked to the gold through the sulphur atom. As PCR proceeds throughannealing, elongation and denaturation, the dielectric constant aroundthe nanoparticle will change dynamically, first with the binding ofsingle strand DNA to primers, then with elongation of the single strandto double strand and finally again with removal of double stranded copyfrom particle surface. The stage of the reaction could be monitoredsimilarly to that of SYBR fluorescence during real time PCR. Using aseparate plasmonic resonance for the probe and heating nanoparticlespecies, the heating nanoparticles will not interfere with measurementat the probe wavelength relative to the resonance of the probenanoparticles. In some embodiments, the probe nanoparticles may have aspherical geometry and be assess by illumination with white light andmeasuring the absorbance using a CCD with a bandpass filter centeredaround the resonance peak. The heating nanoparticle species may be goldnanorods as nanorods have greater extinction coefficients than sphericalparticles and will produce more heating power per unit of laser powerincident upon the nanoparticles.

The wavelength used to initiate plasmon resonance is dependent upon thegeometry of the particle. For example, a 532 nm source as used andexemplified herein may not represent an optimum combination of laserwavelength and particle for some applications and can be modifieddepending on the particles and conditions used. The high cost of lasersin this spectral range would impact upon the uptake of this method, andlight source costs can be significantly reduced by using a lessexpansive laser system or a LED, and choosing and designing thenanoparticles accordingly. Heat transfer can be improved by using aparticle with a large absorption cross-section and hence greatextinction co-efficient. One combination as described herein consists ofa 1 W laser diode at 808 nm and gold nanorods with an absorptivity of5.96×1012M-1 cm-1 at the same wavelength. This takes advantage of asignificant cost reduction and increase in efficiency of heat generationby nanoparticles and offers the potential for multiple nanoparticlesystems that could be easily multiplexed. In such a system, heatingcould be accomplished by a class of nanoparticles with a superiorabsorption cross-section and another class(es) of nanoparticles modifiedusing primers could be used as the probe for the reaction, demonstratingbinding of new amplicon fragments upon the particle surface by changingthe resonant absorbance and spectral position. If a fluorescence systemis required, the additional benefit of changing wavelengths would be toenable the combination with conventional quantitative PCR methods usingintercalating fluorescence dyes such as SYBR green. The laser will besignificantly red-shifted off the fluorescence limiting interference andeliminating issues of dye photobleaching expected with operating a 532nm laser at almost 3 W optical power.

In other variants, the same nanoparticles used to release heat may beused for optical monitoring as well. For example, in the case ofnanorods, the elongated geometry of the nanoparticles results in twodistinct surface plasmon resonances, respectively aligned with thelongitudinal and transversal axes of the nanorod. These two resonancesinteract with light at very distinct wavelengths—for example, thelongitudinal resonance of gold nanorods absorbs light around 808 nm,whereas the transverse resonance absorbs light around 560 nm, and can beused as the monitoring resonance.

With reference to FIG. 5B, there is shown a schematized representationof an apparatus 32 for DNA amplification, extraction or sterilizationwhich includes both a heating module 33 and a monitoring assembly 52. Asexplained above, the heating module 33 includes a thermal block 20 forreceiving the reaction mixture in contact with nanoparticles havingphoto-thermal properties, and a light generating assembly such asheating laser 34 for irradiating the nanoparticles using an activationlight beam 30 such that the nanoparticles release heat sufficient forthe application-related heating purpose. The nanoparticles havingphoto-thermal properties may have a second resonance that can be usedfor probing, or different probe nanoparticles may be put in contact withthe reaction mixture in the thermal block 20 to provide monitoringcapabilities. The monitoring assembly 52 includes a probing light source54 for irradiating the thermal block 20 with a probing light beam havinga wavelength different than a wavelength of the activation light beam,and coordinated either with the second resonance of the heatingnanoparticles or with a resonance of the probe nanoparticles, ifprovided. In the illustrate embodiment, the probing light beam 30outputted by the probing light source 54 is modulated by a pulsingsignal 56 from a locking-amplifier 58. The light of the probing lightbeam 30 is absorbed by the nanoparticles, changing the transmission oflight through the thermal block 20. The corresponding light outputtedfrom the thermal block 20, herein referred to broadly as “return light”50, is measured by a detector 60, such as for example a photodiode. Thephotodiode signal 62 is passed through a pre-amp and the lockingamplifier 58 performs a comparator function to eliminate signal notrelated to the modulation frequency.

The photodiode signal 62 may also be compared to a reference photodiode(not shown) that accounts for laser power fluctuations. The signal fromthis reference photodiode is used to normalise the signal from thesensing photodiode and the result is transmitted as an analog monitoringsignal 64 to the controller 42.

As amplicons are formed the resonance moves changing the absorbance andmoving the plasmon relative to the probe wavelength. Hence the reactioncan be monitored.

The real-time monitoring method described herein presents severalpotential applications, and, apart from sequencing, extend to ultra fastdiagnostic PCR testing utilising the rapid heat transfer enabled bydiffuse nanoscale heaters. It also removes the requirement for capillaryelectrophoresis as a readout methodology. In addition, a reduction intotal volume from microliter volumes used in commercial thermocyclers tonanoliter or picoliter volumes common to chip-level PCR approaches arealso encompassed.

EXAMPLES AND EXPERIMENTAL RESULTS Example 1

Referring to FIGS. 1A, 2, 6 and 7, the results of a first demonstrationof the heating principle described above are shown, In this example, anapparatus such as illustrated in FIG. 2 was used, and the nanoparticleswere put in indirect contact with the reaction mixture, such as shown inFIG. 1A. The PCR reaction mixture was placed within a 0.5 ml tube andcovered with 150 μl of mineral oil to prevent evaporation. The reactionmixture contained: Phusion polymerase (0.02 units/μl), 1×PCR buffer--,nucleotides (10 mM), forward (5′-AACCAGCCCGACTCCTTTG-3′) and reverse(5′-CAGGGGCCAAGTAGAGCATC-3′) primers, bovine serum albumin (10 μg/μl),BHEX plasmid containing the human androgen receptor cDNA (103 ng) anddionized water. Final volume was 25 μl. The glass reaction tube wasimmersed in a 300 μl volume of nanoparticles embodied by goldnanospheres within a 1.5 ml tube and sealed with parafilm.

An activation light beam from a Melles Griot continuous wave laser at awavelength of 532 nm and power of 2.7 W was used to irradiate the doubletube containing the nanoparticles and reaction mixture. An opticalshutter and cooling fan were operated through an Arduino microcontrollerand Labview interface; a 1K thermocouple was inserted into the reactionmixture to record temperature via the microcontroller. The graphicalinterface allowed set temperatures for each stage of the reaction to bedefined as well as the period of time for which each temperature was tobe maintained. Communication between the computer and micro-controllerwas via a USB link. Defined temperatures and times allowed a negativefeedback control mechanism to be instigated where the fan and opticalshutter could be actuated to dynamically alter pulsed excitation ofplasmons dependent upon temperature required and stage of the reactioncycle.

Stability of temperature was demonstrated when using plasmonic heating.Annealing (55.18+/−0.09° C.), elongation (72.03+/−0.13) and denaturingtemperatures (94.09+/−0.1° C.) demonstrated accuracies of 0.1° C.overall. Elongation temperatures are defined for the point where theplateau temperature is reached. FIG. 6 demonstrates the thermocyclingachieved with plasmonic heating; total reaction time for this experimentwas 45 minutes. A reaction was established following the thermal tracein FIG. 6 where DNA was denatured at 94° C. (5 seconds), annealed at 55°C. (20 seconds) and extension or elongation phase occurred at 72° C. (45seconds). The resulting product was separated on a 1.5% agarose gel byelectrophoresis (FIG. 7). Lane 2 contains a positive control produced bya commercial Eppendorf Master thermocycler, lane 3 the negative controland lane 4 the product from the plasmonic thermocycler. Products werevisualized using ethidium bromide and UV gel station, the size markerused was φX174 DNA-HaeIII digest (lane 1).

This example demonstrates that the indirect-contact method can be usedto provide sufficient heating for some target applications. It is to benoted that such embodiments may have the drawback of requiring a largevolume of nanoparticles to heat a small volume of PCR mixture, and maybe an impractical method for potential miniaturisation.

Example 2

Referring to FIGS. 8 to 11, a second example is provided using a directcontact approach, i.e. directly mixing the nanoparticles with thereaction mixture such as for example shown in FIG. 1B. With a particularview to potential PCR applications, the impact of a number of factorswas assessed, such as whether the concentration of nanoparticles issufficient for heating to occur and at what concentration do goldnanoparticles of 60 nm diameter inhibit the polymerase.

First, the potential inhibition of the PCR reaction with goldnanoparticles was investigated in combination with a PCR additive suchas bovine serum albumin (BSA) to prevent polymerase adhering to thenanoparticles. It is likely that the mechanism for inactivation of thepolymerase involves the positively charged active site adsorbing to thesurface through electrostatic interactions with the negatively chargedcitrate capped nanoparticles. This would effectively exclude the bindingof single-stranded DNA. The physical adhesion to the surface of BSAshould create a coating layer, allowing the polymerase to remain free insolution.

Using a dilution series from the stock gold nanoparticle solution (26.3pM), water was replaced with increasing volumes of gold nanoparticles inaqueous solution to yield concentrations ranging from 4.4 pM to 17.9 pMwithin a PCR mixture. Each reaction has an increasing quantity of goldnanoparticles. Reactions were performed upon an Eppendorf thermocyclerand at 4.4 pM no inhibition of the reaction was observed as shown inFIG. 8A. In addition, FIG. 8B shows a measurement of the localisedsurface plasmon absorbance at the concentration of 4.4 pM to demonstratethat the resonance wavelength is unaffected by incorporation into thePCR mixture, as the resonance is still at 532 nm. On the gel it is clearthat the control product in lane 2 matches that of the reaction with 4.4pM of nanoparticles as an addition to the mixture. Subsequentexperimentation demonstrated that the addition of 1.5 ml of 10 ng/ml BSAto the mixture also allowed a greater quantity of nanoparticles to beadded; up to 6.6 pM could be used.

The direct contact heating approach did not initially yield a productfrom the thermocycling or in repeat experiments where low concentrationsof nanoparticles and longer reaction cycles were applied. To resolve thesource of the reaction inhibition an exclusion study was performed basedon three hypotheses. The first hypothesis considered that a 532 nm laserused to excite plasmons also presents the potential for 2 photonabsorbance by thymine residues and the formation of cyclobutylpyrimidine commonly known as a fused base pair that would preventeffective denaturation of double strand DNA and polymerase action. Themechanism is through the absorbance of two photons of longer wavelengthhence lower energy equal to the energy of a single UV photon (˜250-260nm). The second hypothesis was a 2 photon absorbance by aromatic aminoacids (tyrosine, tryptophan, phenylalanine) in the Phusion polymerase.The mechanism in this event would be the generation of free radicaloxygen leading to protein denaturation through excitation of the tripletstate commonly associated with aromatic amino acid fluorescence, but aconsequence of triplet state occupancy is the potential generation ofhighly reactive singlet oxygen. A third hypothesis can be made thatexcludes the potential of singlet oxygen but considers two potentialnanoparticle related effects. A concern was that polymerases, as withother enzymes, are known to interact with gold nanoparticles byelectrostatic adsorption to the particle surface, this would effectivelyblock the active site preventing DNA polymerisation. The secondpotential effect was denaturation of proteins via nanoparticle heatingas has been observed for albumin. This would present a framework forinvestigating the reaction failing in both rapid and conventionalcycling using the contact plasmonic PCR method.

To investigate these three hypotheses a set of reactions was establishedusing both the plasmonic thermocycler and a conventional commercialthermocycler. The PCR mixture, gold concentration, water and enzymequantities are identical to earlier experiments for contact PCR, wherethe gold concentration was 4.4 pM.

TABLE 2 Thermocycling runs used to determine the cause of PCR inhibitionReaction DNA Phusion Addition Lane Product 1M Yes Yes N/A 1 Yes 1L YesYes N/A 6 No 2M No Yes DNA 2 Yes 2L No Yes DNA 3 No 3M Yes No Phusion 5Yes 3L Yes No Phusion 4 Yes

Table 2 shows the reactions established. 1M and 1L are PCR reactions runfor 30 cycles in either the conventional PCR instrument (EppendorfMastercycler or M) or the plasmonic thermocycler (L). 2M and 2L are runfor 15 cycles in either instrument without DNA present to assess theeffect of heat treatment or laser irradiation upon the Phusion enzymerespectively prior to a full 30 cycle run in the Mastercycler to see ifthe reaction will proceed to formation of product. 3M and 3L also aretreated for 15 cycles in the Mastercycler and plasmonic thermocyclerseparately, but with DNA present in the mixture to assess potential ofdamage to DNA prior to conventional amplification. In all cases, if thecomponent of the reaction that is present is damaged, then it willbecome evident as no product will be formed by the conventionalamplification in the Mastercycler following the treatment in theplasmonic thermocycler. The 30 cycle reaction conducted after plasmonicthermocycler pre-treatment was under the following conditions: 98° C.for 30 s hot start, 96° C. for 45 s, 55° C. for 45 s and 72° C. for 45s. The laser was operated at 2.7 W optical power as before. Thetemperature protocol was used consistently between each instrument withrespect to cycling.

The results are indicated in Table 2 and in the gel image in FIG. 9. 1Mprovided the positive control for the experiment and produced product,confirming that the master mixture was made competently. 1L was thenegative control where no template DNA was present and it produced noproduct, confirming no contamination of the master mixture. 2M producedPCR product, removing the possibility of simple heat-relateddenaturation of Phusion, whereas 2L produced no product suggestingpossible laser damage of the enzyme or some other form of enzymedeactivation. 3M and 3L did not initially have any enzyme present duringtheir heat and laser pre-treatment respectively, but primer and templateDNA were present and had enzyme added after. Both reactions werefinished in the conventional thermocycler and produced products. Thisindicated that no DNA damage resulted from laser irradiation in thepresence of gold nanoparticles.

The second possible conclusion is that the enzyme had been deactivatedthrough denaturation or inactivation. As part of the hypothesis for thisexperiment a photochemical route to generating free radicals wasconsidered. It should also be noted that gold nanoparticles have thepotential to form oxygen free radicals, but no evidence for DNA damagecan be demonstrated for the concentration of nanoparticles in thereaction mixture as seen by the successful product formed from reaction3L. It was concluded that the source of reaction failure was linked tothe enzyme; the exact method of inhibition was still unclear asexperiments to quantify singlet oxygen usingtrans-1-(2′-methoxyvinyl)pyrene proved inconclusive.

A simpler explanation was considered than either of the above threehypotheses. The presence of the thermocouple in the reaction mixture hasbeen shown to inhibit polymerases previously, and was solved by theaddition of PEG-8000 to the reaction mixture. The opportunity for volumereduction well in advance of current picoliter systems and the rapidheat exchange over small distances leading to greater reduction inreaction times necessitated a trial of this simple solution. Theaddition of PEG-8000 (0.9% w/v) allowed the demonstration of the contactplasmonic PCR reaction with nanoparticles within the reaction mixture.In addition, a lower denaturation temperature of 90° C. was required asit appears that the nanoparticles may aid denaturation at lowertemperatures. FIG. 10 contains the electrophoresis image of the agarosegel. Lanes 3-5 contain products amplified by plasmonic PCR, lane 2provides the negative control and lane 6 is the positive control using acommercial PCR instrument.

All reactions in this example were performed using taq polymerase.Temperature conditions were 90° C. (30 s), 55° C. (30 s) and 72° C. (30s) until the final 5 cycles where annealing and elongation times wereincreased to 45 seconds. This represents the first demonstration of aPCR reaction driven by nanoparticle plasmonic heaters in direct contactwith DNA and the polymerase.

Careful analysis of the thermocycler's temperature control was performedto provide a comparison to commercial systems and also to characterisethe regulation of temperature. In order to determine maximum heating andcooling rates, a run was performed whereby the solution temperature wasrapidly cycled between 45° C. and 90° C. (FIG. 11). The temperaturerange mirrors the same denaturing and annealing temperatures asperformed by Wheeler et al. (2011, Analyst, 136: 3707-3712) and theirrapid cycling approach. Data were also acquired from a contact PCRexperiment for the purpose of ascertaining temperature stability (FIG.12).

Using the rapid cycling data, heating and cooling rates were calculatedby measuring the difference between successive temperature maxima andminima, then dividing by the time interval between them. The averagerates and sample standard deviations were obtained. FIG. 13 and Table 3detail the results.

TABLE 3 Results of temperature data analysis where standard deviation isa measure of precision Temperature change rates Heating 7.62 ± 0.81° C.s⁻¹ Cooling 3.33 ± 0.24° C. s⁻¹ Temperature stability Denaturation at91° C. 90.87 ± 0.17° C. Annealing at 55° C. 55.10 ± 0.16° C. Elongationat 72° C. 71.92 ± 0.15° C.

The heating and cooling rates obtained are 7.62±0.81° C./second and3.33±0.24° C./second, respectively. FIGS. 1A to 14C indicate that theheating and cooling rates start to stabilise after 10 cycles. Heatingrates initially increase whereas cooling rates decrease. This could bedue to the Eppendorf plastic tube gradually heating up until it reachesa stable temperature. Conversely, one of the fan effects during coolingwould be to create a velocity distribution of the air that is repeatablefrom cycle to cycle, leading to a smaller spread of the cooling ratesrelative to those associated with heating.

Temperature stability of the instrument was also determined using thecontact PCR data presented in FIG. 12. For each PCR step (denaturation,annealing, and elongation) all of the corresponding data points wereaggregated. Averages and sample standard deviations were then computed(FIGS. 14A to 14C and Table 3). The results are 90.87±0.17° C. fordenaturation (91° C. target), 55.10±0.16° C. for annealing (55° C.target), and 71.92±0.15° C. for elongation (72° C. target). The accuracywas determined by computing the fraction of data points that were atmost one bit depth of the analog to digital converter away from thetarget temperature. The accuracy obtained was 22.2% for denaturing,24.8% for annealing, and 26.6% for elongation, where accuracy is definedas the percentage of measurements that exactly hit the definedtemperatures for each stage to within the accuracy of the analog todigital conversion of the measurement system.

FIGS. 14A to 14C demonstrate that the instrument is capable ofmaintaining a defined temperature within stabilities comparable tocommercial instruments. The performance was compared to other PCRinstruments, both open source and commercial. Table 4 summaries thoseinstruments and the plasmonic thermocycler is capable of deliveringsimilar or better stabilities than available instruments (Table 3).

TABLE 4 Comparison of commercially available PCR instruments StabilityRamp Instrument ° C. rate ° C. s⁻¹ Open PCRhttp://openpcr.org/the-machine/ ±0.5 1 Veriti thermocyclerhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/pcr/thermal-cyclers.html±0.5 5 Lightcycler 1536https://www.roche-applied-science.com/sls/rtper/LC1536/index.jsp?id=LC1536_010000N/A 4.8 Thermo scientific arktik thermal cycler http://www.dharmacom.com±0.4 3 Mx3005P QPCR system http://www.genomics.agilent.com  ±0.25 2.5Mastercycler ® pro S http://www.eppendorf.ca ±0.3 6 peqStar thermocyclerhttp://www.peqlab.com/wcms/en/pdf/PEQLAB_peqSTAR2X.pdf ±0.2 5

Of course, numerous modifications could be made to the embodiments abovewithout departing from the scope of the invention as defined in theappended claims.

1-26. (canceled)
 27. A method of amplifying a nucleic acid molecule witha loop-mediated isothermal amplification (LAMP), through bulk heating abiological enzymatic reaction mixture in solution containing a nucleicacid template, a polymerase enzyme, and chemically modifiednanoparticles comprising nanorods of metal, metallic coated organicnanotubes, or a combination thereof, having photo-thermal properties, topromote said LAMP, comprising the steps of: a) irradiating saidchemically modified nanoparticles with an activation light beam from acontinuous wave laser to provide excitation for a period of time toactivate said photo-thermal properties of said chemically modifiednanoparticles, such that said chemically modified nanoparticles releaseheat sufficient to provide said heating to the whole reaction mixture insolution and promote said LAMP.
 28. The method according claim 27,wherein the nanoparticles are selected from the group consisting ofcarbon nanotubes coated with a metal and multiwalled carbon nanotubescoated with or decorated with a metal.
 29. The method according to claim28, wherein the metal is selected from the group consisting of Au, Ag,Pd, Pt, Fe, Cu, Al, and Zn.
 30. The method according to claim 27,wherein said photo-thermal properties comprise a localized plasmonresonance at a surface of the chemically modified nanoparticles, and theactivation light beam has a wavelength corresponding to said localizedplasmon resonance.
 31. The method according to claim 27, wherein thechemically modified nanoparticles are chemically modified by a chemicalcompound that prevents the inhibition of an active site of saidpolymerase enzyme.
 32. The method according to claim 31, wherein thechemical compound that prevents the inhibition of the active site ofsaid polymerase enzyme is polyethylene glycol.
 33. The method accordingto claim 1, wherein the step of irradiating comprises adjusting a powerof said activation light beam to regulate temperature of said biologicalenzymatic reaction mixture in solution through controlled heat releasefrom said chemically modified nanoparticles.
 34. The method according toclaim 1, further comprising a step of monitoring said bulk heating,amplicon production, or both.
 35. The method according to claim 34,wherein the step of monitoring said amplicon production comprisesprobing said chemically modified nanoparticles with a probing lightbeam, having a wavelength different than a wavelength of the activationlight beam and coordinated with an absorption feature of said chemicallymodified nanoparticles spectrally separate from the photo-thermalproperties used to release heat, to measure a change of an opticalproperty of said chemically modified nanoparticles and correlate saidchange of the optical property with a change in said ampliconproduction.
 36. The method according to claim 35, wherein saidchemically modified nanoparticles have an elongated geometry, thewavelength of the activation light beam is coordinated with alongitudinal resonance of the nanoparticles and the wavelength of theprobing light beam is coordinated with a transversal resonance of thenanoparticles.
 37. The method of claim 27, comprising cooling of thereaction mixture after the heating thereof.
 38. The method of claim 27,wherein the reaction mixture in solution comprises a buffer, magnesiumion, a manganese ion, or a combination thereof, deoxynucleosidetriphosphates, and primers.
 39. A method of amplifying a nucleic acidmolecule with a loop-mediated isothermal amplification (LAMP) throughbulk heating a biological enzymatic reaction mixture in solutioncontaining a nucleic acid template, a polymerase enzyme, firstchemically modified nanoparticles comprising nanorods of metal, metalliccoated organic nanotubes, or a combination thereof, having photo-thermalproperties, to release heat and promote said LAMP, and a second set ofnanoparticles having an absorption feature spectrally separate from saidphoto-thermal properties of said first chemically modifiednanoparticles, comprising the steps of: a) irradiating said firstchemically modified nanoparticles with an activation light beam from acontinuous wave laser to provide excitation for a period of time toactivate said photo-thermal properties of said first chemically modifiednanoparticles, such that said first chemically modified nanoparticlesrelease heat sufficient to provide said heating to the whole reactionmixture in solution and promote said LAMP, and b) monitoring said bulkheating, amplicon production, or both, by probing said second set ofnanoparticles with a probing light beam having a wavelength differentthan a wavelength of the activation light beam and coordinated with saidabsorption feature.
 40. The method of claim 39, wherein said second setof nanoparticles is second chemically modified nanoparticles comprisingnanorods of metal, metallic coated organic nanotubes, or a combinationthereof, having photo-thermal properties, and having an absorptionfeature spectrally separate from said photo-thermal properties of saidfirst chemically modified nanoparticles.
 41. The method of claim 27,wherein said nucleic acid template is from a cell, a virus or abacteria.
 42. The method of claim 27, further comprising the step ofextracting said nucleic acid template from a cell, a virus or a bacteriaprior to amplifying said nucleic acid molecule with LAMP.
 43. The methodof claim 41, wherein said step of extracting said nucleic acid templatefrom a cell, a virus or a bacteria comprises irradiating said chemicallymodified nanoparticles with an activation light beam from a continuouswave laser to provide excitation for a period of time to activate saidphoto-thermal properties of said chemically modified nanoparticles, suchthat said chemically modified nanoparticles release heat sufficient forextraction of said nucleic acid template from said cell, virus orbacteria.